Functional Characterization and Localization of a Bacillus ...Functional Characterization and...

Functional Characterization and Localization of a Bacillus subtilisSortase and Its Substrate and Use of This Sortase System ToCovalently Anchor a Heterologous Protein to the B. subtilis Cell Wallfor Surface Display

Pei Xiong Liew,* Christopher L. C. Wang, and Sui-Lam Wong

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis sug-gests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a�-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring ofthis fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inac-tivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were propor-tional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B.subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusionswere distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also ap-peared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium.Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed onthe surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS,47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Charac-terization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system.This system may have various biotechnological applications.

Bacteria possess a wide range of proteins displayed on the cellsurface that interact with molecules and cells in the environ-

ment. To display proteins on the cell surface, several mechanismsare used in Gram-positive bacteria (17). Surface proteins are ei-ther covalently or noncovalently attached to peptidoglycan. Someeven bind noncovalently to secondary cell wall polymers (42).

Sortase is a membrane-bound enzyme that is responsible forthe covalent attachment of proteins to the peptidoglycan of Gram-positive bacteria (39). These wall-anchored surface proteins con-tain two essential elements. First, an N-terminal signal peptide isrequired to direct these proteins to the secretory pathway. Second,a C-terminal cell wall anchoring domain (CWAD) is required forcell wall anchoring. CWADs have three crucial features, which arean LPXTG motif (where X can be any amino acid), a hydrophobictransmembrane domain, and a tail with positively charged aminoacid residues (38, 39).

In Bacillus subtilis, two putative sortases (YhcS and YwpE) andputative substrates (YhcR and YfkN) have been identified bybioinformatic analyses (10, 20, 46). Although YhcR and YfkNhave been identified as an endonuclease (45) and a trifunctionalnucleotidase (8), respectively, conclusive demonstration thatthese proteins are covalently anchored to the cell wall has provento be difficult. This is primarily due to the presence of high levels ofextracellular proteases produced by B. subtilis that degrade thesewall-bound proteins. With the first discovery of the structuralgene encoding sortase in Staphylococcus aureus more than a de-cade ago (40) and the subsequent discovery of many sortases andtheir substrates in many Gram-positive bacteria (46), it is of inter-est to determine whether any sortase and sortase substrate exist inB. subtilis, one of the best characterized Gram-positive bacteria.

In this study, we conclusively demonstrate that yhcS and yhcRencode a sortase and a sortase substrate, respectively. YhcS wasfound to be responsible for anchoring YhcR to the cell wall in acovalent manner. To overcome the significant proteolytic degra-dation of surface proteins, the large nuclease domain (1,085 resi-dues, 118.5 kDa) of YhcR was replaced with a small reporter(�-lactamase) and the fusion protein was produced in an eight-protease-deficient B. subtilis strain (WB800). The amounts of thefusion protein anchored to the cell wall were proportional to thelevels of sortase present in the cell. Interestingly, visualization us-ing an epifluorescence microscope showed that wall-bound re-porters were distributed in a helical fashion, while green fluores-cent protein (GFP)-sortase fusions seem to localize in helical arcsor tracks. To our knowledge, this is the first time that the distri-bution of a sortase in rod-shaped bacteria has been elucidated.Notably, a strategy to quantify the number of wall-bound report-ers in an accurate manner was also developed. This covalent sur-face display system can display not only an enzyme (e.g.,

Received 30 June 2011 Accepted 18 October 2011

Published ahead of print 21 October 2011

Address correspondence to Sui-Lam Wong, [email protected].

* Present address: Snyder Institute of Infection, Immunity and Inflammation,University of Calgary, Calgary, Alberta, Canada.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.05711-11

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�-lactamase) but also has the potential to display giant enzymecomplexes (e.g., cellulosomes) for biofuel conversion.

MATERIALS AND METHODSBacteria and growth conditions. Bacillus subtilis strain WB800 (62) wasused as the expression host for cloning and protein production unlessstated otherwise. In this strain, a total of seven extracellular protease genesand a wall-bound protease gene (wprA) have been deleted. Inactivation ofwprA is essential for the successful display of wall-bound proteins. All B.subtilis cells were propagated at 30°C on tryptose blood agar base (TBAB)plates, and Escherichia coli cells were propagated at 37°C on LB agar plates.For protein production, cells were cultured in superrich medium (SRM)(Bacto tryptose, 25 g/liter; yeast extract, 20 g/liter; dipotassium phosphate,3 g/liter; glucose, 4.5 g/liter; pH 7.5) at 30°C for 14 h and harvested foranalysis. The following antibiotics were used for selection: kanamycin (10�g/ml), spectinomycin (250 �g/ml), ampicillin (75 �g/ml), erythromy-cin (5 �g/ml), and lincomycin (5 �g/ml). B. subtilis transformation wasperformed by the Spizizen competent cell method (55). Table S1 in thesupplemental material lists the bacterial strains used in this study.

Construction of expression vectors and strains. To allow covalentanchoring of the recombinant B. subtilis sortase substrates to the cell wall,an expression vector, pWB980-BLA-CWADYhcR, was constructed (seeFig. S1 in the supplemental material). This vector contains the codingregion for the YhcR cell wall anchoring domain (CWAD) inserted in theexpression vector pWB980-BLA-L56-LytE (9) which produces a�-lactamase (BLA) fusion protein. At the C-terminal end of BLA-L56-LytE is a cell wall binding module derived from LytE, a B. subtilis cell wallhydrolase, for binding to the cell wall in a noncovalent manner. There is a56-amino-acid linker (L56) located between BLA and the LytE cell wallbinding domain. The cell wall binding module of LytE was replaced by theCWAD derived from YhcR (CWADYhcR) by the following process. A438-bp DNA fragment encoding CWADYhcR was amplified by PCR usingB. subtilis WB800 chromosomal DNA as the template and two primers,BSCYHCRF and BSCYHCRB (see Table S2 in the supplemental material).The PCR product was digested with BglII and NheI and inserted intothe BclI/NheI-digested pWB980-BLA-L56-LytE vector to generatepWB980-BLA-CWADYhcR. To produce another fusion protein,BLA-CWADYhcRMO2 (Fig. S1), the expression vector pWB980-BLA-CWADYhcRMO2 was constructed in a two-step process. First, sequenceencoding CWADYhcR was modified to eliminate the termination codonand to introduce a NotI site at the 3= end. This modified sequence wasgenerated by PCR with pWB980-BLA-CWADYhcR as the template andBSSacBF/YHCRCTMB as the primer pair (Table S2). The 1,439-bp PCRproduct was then digested with BstEII and NheI and ligated to the BstEII/NheI-digested pWB980-BLA-CWADYhcR to generate pWB980-BLA-CWADYhcRCTM, which contained a modified 3= end at the coding se-quence of CWADYhcR for the insertion of the monomeric orangefluorescent protein 2 (MO2) gene. In the second stage of construction, theplasmid pBSK-KCMO2ST3 (Table S1), an E. coli Bluescript plasmid car-rying a synthetic gene encoding MO2, was used. This synthetic gene wasordered from Epoch Biolabs (Missouri City, TX). The mo2 nucleotidesequence was redesigned for optimal expression in B. subtilis based on thepreviously reported protein sequence for MO2 (51). pBSK-KCMO2ST3was double digested with NotI and NheI to release the mo2 insert, whichwas then inserted into the NotI/NheI double digested pWB980-BLA-CWADYhcRCTM to generate pWB980-BLA-CWADYhcRMO2. This vectorallows the production of a fusion protein with �-lactamase at theN-terminal region, the CWADYhcR domain in the middle, and MO2 at theC-terminal end.

Plasmid pWB980-YhcS is an expression vector for the production ofthe wild-type full-length sortase, YhcS. The 716-bp full-length yhcS wasgenerated by PCR amplification of the B. subtilis WB800 chromosomalDNA with primers BSYHCSF and BSYHCSB (see Table S2 in the supple-mental material). The PCR product was digested with KpnI and NheI andinserted into KpnI/NheI-digested pWB980 (61) to generate pWB980-

YhcS. For the simultaneous production of both YhcS and BLA-CWADYhcRMO2 in the same expression host, a pWB980-pE18 binary vec-tor system was used (61). One plasmid (pWB980-BLA-CWADYhcRMO2,kanamycin resistant) overproduces BLA-CWADYhcRMO2. The other plas-mid (pE18-YhcS, erythromycin resistant) overproduces YhcS. These twoplasmids can coexist in the same B. subtilis host. To construct pE18-YhcS,yhcS was subcloned into pE18-P43 (61) by first cutting pWB980-YhcSwith NheI, blunt ended using T4 DNA polymerase I and subsequently cutwith BlpI to release the insert carrying yhcS. This fragment was insertedinto SmaI/BlpI-digested pE18-P43 to generate pE18-YhcS.

YhcS has an N-terminal transmembrane segment followed by a sortasecatalytic domain. To purify the non-membrane-bound form of sortasefor injection into mice to obtain antisortase antibodies, a glutathioneS-transferase (GST) sortase fusion construct (GST-YhcSNM) was made.YhcSNM is a truncated version of YhcS with its N-terminal transmem-brane segment deleted (NM designates the non-membrane-bound ver-sion). GST-YhcSNM is soluble and can be affinity purified. PlasmidpGEX2T-YhcSNM was made in a two-step process. The first step is togenerate pWB980-YhcSNM. A PCR product encoding YhcS was gener-ated by using B. subtilis WB800 genomic DNA as the template andBSYHCSF and BSYHCSB (see Table S2 in the supplemental material) asthe primers. This fragment was digested with both ClaI and NheI andinserted into the ClaI/NheI-digested pWB980-XylA. This generatespWB980-YhcSNM. In the second step, the DNA fragment encodingYhcSNM was amplified by PCR using pWB980-YhcSNM as the templateand yhcSNMfrGSTF and yhcSNMfrGSTR as primers (Table S2). ThepGEX2T plasmid and the PCR product (647 bp) were digested withBamHI and NheI. The PCR product was then inserted into pGEX2T togenerate pGEX2T-YhcSNM. pGEX2T-YhcSNM was transformed into E.coli DH5� for protein production.

Generation of a sortase knockout mutant (WB800Srt�) using theCre/lox- and PCR-based method. B. subtilis WB800Srt� was constructedby the replacement of yhcS in strain WB800 with an antibiotic cassetteusing a genome engineering method developed by Yan et al. (63). Briefly,the spectinomycin resistance (Spcr) cassette (1,178 bp) was amplifiedfrom vector p7S6 (Bacillus Genetic Stock Center) (Table S1) with primersLox71F and Lox66R (see Table S2 in the supplemental material). Twoprimer pairs, 1kbyhcRF/1kbyhcRR (front flanking region) and1kbyhcTF/1kbyhcTR (back flanking region), were used to amplify two1-kb DNA fragments flanking the upstream and downstream yhcS se-quence, respectively. Extensions of 21 nucleotides that were complemen-tary to the 5= and 3= ends of the amplified Spcr cassette were added to the5= ends of both the reverse and forward primers of the upstream anddownstream flanking regions. All three fragments were amplified usingVent polymerase, and the products were used for a two-step PCR fusionamplification. Step A (10 cycles) included 73 �l water, 10 �l ThermoPolbuffer (10�), 10 �l deoxynucleoside triphosphate (dNTP) mix (2.5 mMeach), 2 �l of MgSO4 (100�), 1 �l (100 ng) of the upstream flankingfragment, 2 �l (200 ng) of marker cassette fragment, 1 �l (100 ng) of thedownstream flanking fragment, and 1 �l of Vent polymerase. Step B (30cycles) included 73 �l water, 10 �l ThermoPol buffer (10�), 10 �l dNTPmix, 1 �l MgSO4 (100�), 2 �l (10 mM) forward primer of the upstreamflanking fragment, 1 �l (10 mM) reverse primer of the downstream flank-ing fragment, 1 �l of the unpurified PCR product from step A, and 1 �l ofVent polymerase. The resulting PCR product was gel purified and trans-formed into B. subtilis WB800. The transformants were selected on TBABplates containing 250 �g/ml spectinomycin. The successful generation ofthe yhcS knockout strains was confirmed by PCR screening of the specti-nomycin-resistant transformants. One knockout strain (WB800Srt�) waskept for further studies. To remove the antibiotic Spcr marker, pTSC(Bacillus Genetic Stock Center) (see Table S1 in the supplemental mate-rial) carrying a constitutively expressed Cre recombinase gene was intro-duced into the yhcS knockout strain via transformation. Erythromycin-resistant transformants were selected. These transformants were thengrown on antibiotic-free LB liquid medium for 16 h and plated on LB

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plates. The removal of the Spcr marker was confirmed by the inability ofthese cells to grow on TBAB plates containing 250 �g/ml spectinomycin.To remove the pTSC plasmid, the cells were grown overnight in LB with-out erythromycin at 51°C for 16 h, as pTSC has a temperature-sensitivereplicon and is unable to replicate at 51°C. The resulting erythromycin-sensitive WB800Srt� strain was used for the construction of WB800Srt�

amyE::pxyl-gfp-yhcS.Construction of WB800Srt� amyE::pxyl-gfp-yhcS. To construct a

green fluorescent protein-sortase (GFP-YhcS) fusion strain, pSG1729 (5)was used. This plasmid was obtained from the Bacillus Genetic StockCenter. The full-length sortase gene yhcS was PCR amplified with theprimer pairs GFPYHCSF and GFPYHCSB (see Table S2 in the supple-mental material) using pWB980-YhcS as the template to generate a695-bp PCR product which was subsequently digested by both BamHIand NheI and subcloned into the BamHI- and NheI-digested pSG1729.The resulting clone contains yhcS inserted in the positive orientationdownstream of the green fluorescent protein (GFP) sequence and wasdesignated pSG1729-GFP-YhcS. The mut1 variant (64) of the green fluo-rescent protein gene was fused to the N-terminal end of YhcS at the genelevel, and the resulting gfp-yhcS gene was placed under the control of axylose-inducible promoter (pxyl) of the pSG1729 plasmid that replicatesonly in E. coli. GFP was fused to the N terminus of YhcS to preserve theproper topology and avoid the secretion of GFP. GFP is not normallyfunctional when translocated to the extracellular side of the membrane inbacteria (23, 41).

The pSG1729 plasmid contains the 5= and 3= ends of the B. subtilisamyE sequence for integration into the B. subtilis amyE locus via a double-crossover event and a spectinomycin antibiotic marker for selection. Ashort flexible linker (14 amino acids) between GFP and YhcS was includedwith the intention to separate the two domains and to allow bothdomains to fold independently. Plasmid pSG1729-GFP-YhcS was in-tegrated into the genome of the spectinomycin-sensitive WB800Srt�

strain to generate WB800Srt� amyE::pxyl-gfp-yhcS containing a singlecopy of the inducible GFP-sortase fusion gene. The integration ofpxyl-gfp-yhcS into the amyE locus was confirmed by PCR with primersdiGFPyhcSMR1 and diGFPyhcSMR2 (see Table S2 in the supplemen-tal material). The sortase knockout strain WB800Srt� was chosen toensure that the localization signal of GFP-YhcS was not displaced bythe presence of wild-type YhcS.

Release of wall-bound forms of BLA-CWADYhcR and BLA-CWADYhcRMO2 by lysozyme. Profiling of sortase substrates was exam-ined by subjecting B. subtilis WB800(pWB980-BLA-CWADYhcR) (forBLA-CWADYhcR) and WB800(pWB980-BLA-CWADYhcRMO2) (for BLA-CWADYhrRMO2) to lysozyme digestion. Washed cells were resuspended in150 �l SET buffer (20% sucrose, 50 mM Tris, 50 mM EDTA [pH 7.6]).Lysozyme and phenylmethylsulfonyl fluoride (PMSF) were added to thecell suspensions to a final concentration of 8 mg/ml and 1 mM, respec-tively. Samples were incubated at 37°C for 30 min. Protoplast formationwas confirmed by using a phase-contrast microscope. Protoplasts wereseparated from the digested cell wall by centrifugation using a microcen-trifuge at 5,400 � g for 7 min. The resulting protoplast pellet was resus-pended with 150 �l of SET buffer. Both the protoplast and cell wall frac-tions were used for Western blotting.

Immunofluorescence microscopy. Localization of wall-bound BLA-CWADYhcRMO2 on the bacterial surface was examined by using an epiflu-orescence microscope as previously described (9) with the followingchanges. Briefly, cells cultured overnight in SRM were harvested and fixedwith 4% paraformaldehyde for 30 min at 4°C after they were washed withphosphate-buffered saline (PBS). Cell samples were then blocked with 1%(vol/vol) bovine serum albumin (BSA) for 1 h before they were incubatedwith rabbit primary antibodies against �-lactamase in PBS in a microcen-trifuge tube with gentle agitation for 2 h. The cells were washed three timeswith PBS containing 0.1% Tween 20 (vol/vol) (5 min per wash) beforebinding with Alexa Fluor 488-conjugated goat anti-rabbit antibodies andAlexa Fluor 594-conjugated wheat germ agglutinin (WGA) for another 2

h in microcentrifuge tubes with gentle agitation. WGA bindsN-acetylglucosamine in the peptidoglycan. The cells were washed withPBS containing 0.1% Tween 20 again before 4=,6=-diamidino-2-phenylindole (DAPI), a fluorescent dye for DNA, was added. The cellswere then spotted onto glass microscope slides, and SlowFade antifadereagent was subsequently added to prevent photobleaching. The stainedcells were visualized.

As the excitation and emission wavelengths of Alexa Fluor 488 coin-cide with those of GFP, it would not be possible to use the same secondaryantibodies for the colocalization studies of BLA-CWADYhcRMO2 withGFP-YhcS. B. subtilis WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) was probed with Alexa Fluor 568-conjugated rabbit anti-FLA antibodies after the binding of the primary antibodies. The cells werewashed again and spotted onto glass microscope slides for visualizationusing the fluorescence microscope. Optical z-stacks were taken. Imageswere saved as ZVI files for deconvolution software processing. Deconvo-lution was applied to images using Huygen’s deconvolution software (Sci-entific Volume Imaging, Hilversum, Netherlands). The Carl Zeiss Axio-imager Z1 fluorescence compound microscope was used for imaging. TheZeiss 100� objective was used for image capture.

Other methods. Cell wall from a late-log-phase B. subtilis culture wasisolated as described previously (9). Purified GST-YhcSNM was used asthe antigen to generate polyclonal antibodies in mice according to thepreviously published procedures (58). GST-YhcSNM was purified usingglutathione-Sepharose columns as recommended by the manufacturer.Rabbit antibodies against �-lactamase generated previously (59) wereused in this study. The numbers of wall-bound BLA-CWADYhcRMO2 in B.subtilis WB800(pWB980-BLA-CWADYhcRMO2) and WB800(pE18-YhcS,pWB980-BLA-CWADYhcRMO2) were estimated by Western blotting usinganti-BLA antibodies as previously described (9) with the followingchanges. Known numbers of bacterial cells were treated with lysozymebefore separating the digested cell wall from protoplasts by centrifugation.The soluble supernatant containing the digested cell wall was separated bySDS-PAGE for Western blotting. The activity of BLA was assayed as pre-viously described (9) with the following modifications. The assayused 7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethyl-amino-phenylazo)pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC) as the sub-strate and was carried out using 96-well microtiter plates. The total vol-ume of each assay mix (consisting of PADAC and PBS) per well was 100�l. The amount of PBS containing PADAC (PADAC-PBS) was adjustedsuch that the initial optical density at 595 nm (OD595) observed was 1.00(�0.05) before the start of the assay. Washed cells that were resuspendedin PBS were pipetted to the assay mixture to begin the assay, and readingsat OD595 were recorded at 1-min intervals for a total of 10 min. The totalvolume of the cell samples added to PADAC-PBS for the assay was ad-justed such that the rate of product formation was linear. Enzymatic ac-tivities were calculated using the molar extinction coefficient of 52,700M�1 cm�1.

RESULTSDesign, production, and characterization of BLA-CWADYhcR

and BLA-CWADYhcRMO2. To demonstrate the anchoring of theputative sortase substrate YhcR to the cell wall using the B. subtilisendogenous sortase, a �-lactamase fusion (BLA-CWADYhcR) wasconstructed. This fusion can be divided into three parts. TheN-terminal portion is the �-lactamase reporter. Its C-terminalend consists of the cell wall anchoring domain derived from YhcR(CWADYhcR). This domain comprises the LPDTS sorting motif, atransmembrane segment, and a positively charged tail (Fig. 1A).Between these two domains is a 152-amino-acid linker region.The first 50 amino acids form a designer linker which is composedof mainly glycine, proline, serine, and threonine residues. It is thenfollowed by 102 amino acids that are naturally present upstream ofthe LPDTS sorting motif of CWADYhcR. Since a linker of �123

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FIG 1 Rational design of the reporter fusions, BLA-CWADYhcR and BLA-CWADYhcRMO2, and their production. (A) Illustration depicting the construction andproperties of the BLA-CWADYhcR reporter fusion. The reporter �-lactamase (BLA) was fused to the cell wall anchoring domain of YhcR at the gene level. Theprecursor protein is directed to the Sec-based secretory pathway by the B. subtilis SacB signal peptide (3 kDa). The SacB signal peptide (SP) is removedduring/after translocation and the membrane-bound form (M) of the fusion is recognized by sortase. Sortase (YhcS) is likely to cleave between the threonine andserine residues of the LPDTS motif and allows anchoring of BLA-CWADYhcR to peptidoglycan to generate the wall-bound form (W) of the reporter. Thetransmembrane segment (TM) and the positively charged C-terminal tail in the cell wall anchoring domain (�) are indicated. In the linker region, the50-amino-acid designer linker (broken line) and the 102-amino-acid linker naturally present in YhcR (solid black line) are indicated. (B) Illustration depictingthe addition of the monomeric orange fluorescent protein 2 (MO2) to generate the BLA-CWADYhcRMO2 reporter fusion. MO2 was introduced into theC-terminal end of BLA-CWADYhcR. With this addition, the transmembrane segment and the positively charged tail of CWADYhcR would acquire a 28.1-kDaMO2 which would shift the membrane-bound form (M) of BLA-CWADYhcRMO2 to a higher-molecular-mass position in SDS-polyacrylamide gels. In contrast,

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amino acids is sufficient to penetrate through the thick B. subtiliscell wall and project the reporter to the cell surface (44), this 152-amino-acid sequence should be long enough for its function as alinker.

Cell wall sorting of BLA-CWADYhcR was examined by two ap-proaches. The first approach was Western blot analysis (with anti-BLA antibodies) of the lysozyme-treated B. subtilis WB800(pWB980-BLA-CWADYhcR) (Fig. 1D) with the nontreated WB800(pWB980-BLA-CWADYhcR) (Fig. 1C) as the negative control. A total of threeforms (the full-length precursor, the membrane-bound interme-diate, and the wall-bound form) of BLA-CWADYhcR (Fig. 1A)would be generated during the sorting process. The wall-boundform of BLA-CWADYhcR was present in the supernatant fractionof the lysozyme-treated sample (Fig. 1D, lane 1) and was absent inthe untreated control (Fig. 1C, lane 1). The membrane-bound andprecursor forms of BLA-CWADYhcR should be present in the pro-toplast fraction (Fig. 1C and D). Since the three forms of proteinhad relatively similar molecular masses (Fig. 1A), it was not asurprise to observe that the mobility of the wall-bound form ofBLA-CWADYhcR was similar to that of the membrane-boundform and/or the precursor form of BLA-CWADYhcR (Fig. 1D,lanes 1 and 2). This property can impose a problem in the quan-tification of the wall-bound form of BLA-CWADYhcR. Any lysis ofthe protoplasts during the lysozyme treatment would release themembrane/precursor forms of BLA-CWADYhcR to the superna-tant fraction. Consequently, the band intensity observed in thesupernatant fraction might not accurately reflect the quantity ofthe wall-bound form of BLA-CWADYhcR released from the ly-sozyme treatment. To address this concern, a 28-kDa monomericorange fluorescent protein 2 (MO2) was added to the C-terminalend of CWADYhcR to generate BLA-CWADYhcRMO2 (Fig. 1B). Af-ter the sortase-mediated processing, the wall-bound form of BLA-CWADYhcRMO2 would have a much smaller molecular mass withreference to both the membrane and precursor forms.

Analysis of the lysozyme-treated samples of B. subtilisWB800(pWB980-BLA-CWADYhcRMO2) indicated that the wall-bound form of BLA-CWADYhcRMO2 was present in the superna-tant fraction (Fig. 1D, lane 3, arrow) in small quantities. A form ofBLA-CWADYhcRMO2 with higher molecular weight (Fig. 1D, lane3, open circle) was also detected in the supernatant fraction. Ob-servation of this higher-molecular-weight form in the supernatantwas likely due to low levels of cell lysis occurring during lysozymetreatment. Without the ability to separate the membrane-boundform from the wall-bound form, the amount of the wall-boundform may easily be overestimated by five times or more. Together,

these results suggest that BLA-CWADYhcRMO2 was covalently an-chored to the B. subtilis cell wall. A discrepancy between the ex-pected and observed molecular weights of BLA-CWADYhcR andBLA-CWADYhcRMO2 was observed. This was due to the presenceof the unstructured designer linker, which results in a slower mi-gration of proteins in an electrophoretic run (9).

To unambiguously confirm the covalent anchoring of BLA-CWADYhcR and BLA-CWADYhcRMO2 to the peptidoglycan, a sec-ond approach was taken. The cell wall fractions of B. subtilisWB800(pWB980-BLA-CWADYhcR) and WB800(pWB980-BLA-CWADYhcRMO2) were prepared and boiled in the presence of SDSto remove any noncovalent wall-bound proteins. Western blotanalysis reveal the presence of the 64-kDa reporter fusion inthe cell wall fractions prepared from WB800(pWB980-BLA-CWADYhcR) and WB800(pWB980-BLA-CWADYhcRMO2) (Fig. 2,lanes 2 and 3). The protein bands with molecular masses greaterthan 64 kDa were likely the wall-bound proteins linked to the cellwall fragments which were not completely digested by lysozyme.The presence of deacetylated glucosamine (2) and attachment ofteichoic acid and covalently linked BLA-CWADYhcR/BLA-CWADYhcRMO2 in B. subtilis peptidoglycan can potentially pre-vent the complete digestion of the cell wall by lysozyme. The cellwall from the negative-control strain WB800(pWB980) showedno detectable �-lactamase fusion bands (Fig. 2, lane 1). Cell wallsamples that were not treated with lysozyme also did not show any�-lactamase bands (Fig. 2, lanes 7 to 9).

Cell wall anchoring of BLA-CWADYhcRMO2 is mediated bythe YhcS sortase. Since yhcS (10, 20, 46) encodes a putative sortaseand is located next to yhcR in the B. subtilis genome, it is of interestto determine whether YhcS is responsible for anchoring BLA-CWADYhcRMO2 to the cell wall. A yhcS knockout strain(WB800Srt�) was constructed (see Fig. S2 in the supplementalmaterial) and confirmed by genomic PCR analysis (data notshown). The effect of knocking out the yhcS gene in theWB800Srt� strain on cell wall protein anchoring was examined incomparison to the positive-control strain by Western blotting. Awall-bound form of BLA-CWADYhcRMO2 was observed in B. sub-tilis WB800(pWB980-BLA-CWADYhcRMO2) (Fig. 3, lane 3, ar-row). This band was not detected in B. subtilis WB800Srt�

(pWB980-BLA-CWADYhcRMO2) (Fig. 3, lane 1, arrow). Similarfindings were observed using purified cell wall fractions fromthese strains (Fig. 2, lane 3 versus lane 5). Loading 10 timesmore purified cell wall prepared from WB800Srt�

(pWB980-BLA-CWADYhcRMO2) still did not allow the detectionof the wall-bound BLA-CWADYhcRMO2 (Fig. 2, lane 5). In con-

the wall-bound form (W) of BLA-CWADYhcRMO2 would have the same molecular mass as the initial BLA-CWADYhcR construct. (C) Western blot analysis of thecellular distribution of BLA fusions from B. subtilis without lysozyme treatment (�). B. subtilis strains WB800(pWB980-BLA-CWADYhcR) (lanes 1 and 2) andWB800(pWB980-BLA-CWADYhcRMO2) (lanes 3 and 4) were analyzed. A centrifugation step was performed to separate the soluble and cell fractions beforeelectrophoresis and immunoblotting. The soluble supernatant fraction (S) and the whole-cell pellet fraction (P) are indicated above the gel. The amounts ofsamples loaded were normalized to cell density. Rabbit anti-BLA was used to probe for the reporter fusion with a 1:2,000 dilution. The positions of molecularmass markers (in kilodaltons) are indicated to the left of the gel. (D) Western blot analysis of the cellular distribution of BLA fusions from B. subtilis with thelysozyme treatment. Strains WB800(pWB980-BLA-CWADYhcR) (lanes 1 and 2) and WB800(pWB980-BLA-CWADYhcRMO2) (lanes 3 and 4) were treated withlysozyme (�) to release the wall-bound reporters. A centrifugation step was performed to separate wall-bound and membrane-bound forms before electropho-resis and immunoblotting. The soluble supernatant fraction (S) and the protoplast fraction (P) are indicated above the gel. The membrane-bound form (M)(open circle) and the wall-bound form (W) (arrow) of BLA-CWADYhcRMO2 are shown. The amounts of samples loaded were normalized to cell density. Rabbitanti-BLA was used to probe for the reporter fusion with a 1:2,000 dilution. (E) Predicted and observed molecular masses of BLA-CWADYhcR and BLA-CWADYhcRMO2. The observed molecular masses of the fusion proteins on SDS-polyacrylamide gels are constantly larger than the predicted values because of thepresence of the unstructured designer linker region. A detailed explanation is provided in reference 9. The precursor and membrane-bound forms of the fusionproteins have similar molecular masses. The precursors may comigrate with the membrane-bound form of the fusions on SDS-polyacrylamide gels. Asterisksindicate the observed molecular masses of the precursors if they exist.




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trast, the ability to anchor BLA-CWADYhcRMO2 covalently to thecell wall (Fig. 2, lane 4; Fig. 3, square) could be restored by intro-ducing yhcS back into the Srt� strain [i.e., WB800Srt�(pE18-YhcS, pWB980-BLA-CWADYhcRMO2)]. No wall-bound BLA-CWADYhcRMO2 could be detected from WB800Srt�(pE18,pWB980-BLA-CWADYhcRMO2) (data not shown). These data in-dicate that knocking out yhcS abrogates the sorting of BLA-CWADYhcRMO2 to the cell wall and that YhcS is the sortase respon-sible for anchoring BLA-CWADYhcRMO2 to the peptidoglycan.Furthermore, yhcS is not an essential gene required for the survivalof B. subtilis when cells are cultivated in the superrich medium.

Sortase levels determine the amounts of BLA-CWADYhcRMO2

anchored to the cell wall. The physiological level of YhcS in B.

subtilis WB800 was very low (Fig. 4A, lane 2). Even though thesample was concentrated 6 times, no YhcS could be detected byWestern blotting. Introducing the pE18-YhcS plasmid into B. sub-tilis strains WB800, WB800(pWB980-BLA-CWADYhcRMO2), andWB800Srt� resulted in overproduction of YhcS in these strains.Higher levels of cellular YhcS led to increases in the amounts of thewall-bound BLA-CWADYhcRMO2 (Fig. 4B, arrows; see Fig. S4C inthe supplemental material) in WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2). These results show that the sortase levelacts as a “bottleneck” step which limits the number of BLA-CWADYhcRMO2 that can be anchored to peptidoglycan.

Immunofluorescence visualization of BLA-CWADYhcRMO2.The surface accessibility and distribution profile of wall-bound BLA-CWADYhcRMO2 were examined by using immunofluorescence mi-croscopy. B. subtilis WB800(pWB980-BLA-CWADYhcRMO2) wasstained with a DNA-specific fluorescent dye (DAPI [blue]), a pep-tidoglycan binding agent, Alexa Fluor 594-conjugated wheat germagglutinin (WGA) (red) and rabbit antibodies against BLA(probed with Alexa Fluor 488-conjugated secondary antibodies[green]) (Fig. 5A, panels a to d). Superimposing the three differentcolored images suggests that BLA-CWADYhcRMO2 was displayedon the cell surface (Fig. 5A, panels e to h).

Interestingly, the images reveal that BLA molecules were dis-tributed nonuniformly in a helical manner along the lateral wall.To acquire clear fluorescence signals and sharper images, z-stacksthat were transverse to the short axis of the cell were obtained, anda deconvolution algorithm was applied (Fig. 5B). Examining in-dividual image sections obtained under different z-planes for thegreen channel (Fig. 5C) reveals a helical pattern of localization ofBLA-CWADYhcRMO2 along the cell wall (5, 14, 49). These helicestracked along the entire cell length and were tilted in comparisonto the longitudinal axis of the cell (Fig. 5C). The nonuniformdistribution of BLA-CWADYhcRMO2 was also observed in B. sub-tilis WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2) (datanot shown).

FIG 2 Western blot analysis of the covalently linked wall-bound �-lactamasefrom the purified B. subtilis cell wall preparations. Purified cell wall samplesfrom B. subtilis strains WB800(pWB980) (control [C]), WB800(pWB980-BLA-CWADYhcR) (R), WB800(pWB980-BLA-CWADYhcRMO2)(RM), WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2) (sortase overproduction [S�]),and WB800Srt�(pWB980-BLA-CWADYhcRMO2) (sortase knockout [SKO])were probed with anti-BLA antibodies in the Western blot analysis. Samples inlanes 1 to 5 were treated with lysozyme (�) to release BLA-CWADYhcR, andsamples in lanes 7 to 10 were not treated with lysozyme (�). Lane 1, cell wallfrom B. subtilis WB800(pWB980) which has the expression vector without anyinserts; lane 6, lysozyme in buffer (L). The samples in both lanes 1 and 6 arenegative controls. The filled arrowhead marks the expected wall-bound formof BLA. The open arrowheads mark the wall-bound BLA anchored to theincompletely digested cell wall. The amounts of cell wall loaded were normal-ized against the cell density. The only exception is the sample from strainWB800Srt�(pWB980-BLA-CWADYhcRMO2). The amount of this sample(SKO) loaded is 10 times more than the normalized amount of cell wall fromother samples.

FIG 3 Knockout of yhcS abrogates cell wall anchoring of BLA-CWADYhcRMO2. Western blot analysis of the wall-bound BLA-CWADYhcRMO2

was performed. B. subtilis strains WB800Srt�(pWB980-BLA-CWADYhcRMO2), WB800(pWB980-BLA-CWADYhcRMO2), and WB800Srt�

(pE18-YhcS, pWB980-BLA-CWADYhcRMO2) were treated with lysozyme.BLA-CWADYhcRMO2 molecules produced from strain WB800(pWB980-BLA-CWADYhcRMO2) were used as the positive control. S, the supernatant fraction;P, the protoplast pellet. The membrane-bound form of BLA-CWADYhcRMO2 ismarked by the open circle. The arrow in lane 1 highlights the loss of thewall-bound form of BLA-CWADYhcRMO2 in strain WB800Srt�(pWB980-BLA-CWADYhcRMO2). The square depicts the presence of wall-bound BLA-CWADYhcRMO2 with the rescue of sortase activity. The amounts of samplesloaded were normalized to cell density. Rabbit anti-BLA was used to probe forthe reporter fusion with a 1:2,000 dilution.

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In the control strain, WB800(pWB980), which was stainedsimilarly, no binding of the anti-BLA antibodies was observed(Fig. 5D). Furthermore, WB800(pWB980-BLA-CWADYhcRMO2)cells probed only with Alexa Fluor 488-conjugated rabbit anti-BLA antibodies also did not show any fluorescence under thegreen, red, or blue channels used (Fig. 5E). All these data demon-strate that (i) BLA-CWADYhcRMO2 molecules were surface acces-sible and (ii) BLA-CWADYhcRMO2 molecules distribute in a helicalmanner.

Production of GFP-YhcS and its biological activity. SinceBLA-CWADYhcRMO2 distributed helically on the cell wall, it wouldbe of great interest to determine the distribution of YhcS in B. subtilis.A GFP-YhcS strain (WB800Srt� amyE::pxyl-gfp-yhcS) was con-structed. Production of GFP-YhcS in this strain could be observed(see Fig. S3 in the supplemental material) only under induction con-ditions. In both strains (WB800Srt� amyE::pxyl-gfp-yhcS with orwithout pWB980-BLA-CWADYhcRMO2), induction with xylose didnot reveal any differences in the GFP-YhcS level when the xylose levelwas varied from 0.5% to 2.0% (Fig. S3B and S3C, lanes 2 to 5 and 7 to10). However, the amount of GFP-YhcS seemed to be slightlylower in strain WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) than in strain WB800Srt� amyE::pxyl-gfp-yhcS. Thisis not unexpected, since a portion of the cellular energy is consumedfor the production of the BLA fusion in WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2).

To confirm that YhcS is functional in a GFP fusion format,sorting of BLA-CWADYhcRMO2 to the cell wall in B. subtilisWB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2)was examined. The amount of wall-bound BLA-CWADYhcRMO2

produced from WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) was even more than that produced fromWB800(pWB980-BLA-CWADYhcRMO2) (Fig. 4C, lane 2 versuslane 1). This demonstrates that some, if not all, of the GFP-YhcSfusion proteins are active and able to anchor BLA-CWADYhcRMO2

to the peptidoglycan. Higher levels of wall-bound BLA-CWADYhcRMO2 produced from WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) suggests that the xylose pro-moter under induction conditions is stronger than the naturalyhcS promoter.

Distribution of the GFP-YhcS sortase in B. subtilis. To inves-tigate the spatial distribution of GFP-YhcS, xylose-inducedWB800Srt� amyE::pxyl-gfp-yhcS cells were examined by using epi-fluorescence microscopy. The fluorescent foci of GFP-YhcS wereobserved to be nonuniformly distributed around the cell periph-ery in the form of patches or short arcs (Fig. 6). The fluorescencesignals in these cells were much more diffuse than those observedfor the wall-bound BLA-CWADYhcRMO2.

To further investigate whether the arcs would track laterallyalong the membrane, the fluorescent pattern of GFP-YhcS wasobserved in stacks of optical sections. Two different cells are pre-sented here to illustrate the different helices observed in B. subtilisWB800Srt� amyE::pxyl-gfp-yhcS (Fig. 6B and C). In some cells,some of these structures were observed to consist of tracks thatresemble intertwining ribbons (Fig. 6B, panel c) while in othercells, discrete arcs were seen to localize to the cell periphery indifferent planes (Fig. 6C, panels c to e). The optical sectionsthrough successive planes reveal that these helical tracks traverseabout half of a helical turn (Fig. 6C, white arrows). These heliceswere observed to be tilted relative to the long axis of the cell.Although Fig. 6B reveals an intersecting helix, complete helices

were rarely detected in most cells. In distinct contrast, the controlproducing only GFP (WB800Srt� amyE::pxyl-gfp) gives rise to auniform green fluorescence located throughout the cell’s cyto-plasm (Fig. 6D). This indicates that GFP by itself does not localizeto the cell periphery.

Interaction between BLA-CWADYhcRMO2 and GFP-YhcS isnecessary for sorting BLA-CWADYhcRMO2 to the cell surface.Therefore, it is logical to predict that there should be some degreeof transient colocalization of BLA-CWADYhcRMO2 with GFP-YhcS. Xylose-induced WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) cells were probed with anti-BLA antibodiesfor fluorescence visualization studies (Fig. 7A). Some of the BLA-CWADYhcRMO2 signals overlapped with a short segment of a GFP-YhcS arc (Fig. 7B, red arrows). The observed colocalized fluores-cent signals from both proteins suggest that some GFP-YhcS andBLA-CWADYhcRMO2 are in close proximity to one another. Whenviewing progressively along the z-stack plane (i.e., downwardsthrough the cell) (Fig. 7B, panels a to f), colocalization of the twoproteins appears to occur progressively (red arrow 1 in Fig. 7B inpanel a is followed by red arrows 2 and 3 in panels b and c beforethe appearance of red arrow 4 in panels d and e). The appearanceof these colocalization spots is consistent with the idea that BLA-CWADYhcRMO2 molecules are inserted into the cell wall followingthe GFP-YhcS path at a given moment.

Comparison of sortase production levels in wild-type and re-combinant B. subtilis strains. To systematically compare the sor-tase levels of these strains, whole cells from B. subtilis WB800,WB800Srt�, WB800(pE18-YhcS), WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2), WB800Srt�(pE18-YhcS), WB800Srt� amyE::pxyl-gfp-yhcS, and WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) were used in this study. Strains WB800Srt� andWB800 were concentrated six times based on cell density measure-ment for this study.

No sortase was detected in B. subtilis WB800Srt� (Fig. 4A, lane1). Unfortunately, no sortase could be detected from strainWB800 either (Fig. 4A, lane 2). Despite repeated efforts to useconcentrated cell samples (six times more concentrated) (forWB800Srt�and WB800) and more anti-YhcS antibodies, immu-noblotting did not give any detectable signal for these samples.This is likely attributed to a low expression level of yhcS in strainWB800. In comparison, significantly larger amounts of sortasewere found in all three strains which had the pE18-YhcS plasmid[i.e., WB800(pE18-YhcS), WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2), and WB800Srt�(pE18-YhcS)]. The levels of sor-tase detected from these three strains were comparable (Fig. 4A,lanes 3 to 5, a 21-kDa protein band which matches the expectedsize of YhcS). Interestingly, induction of the single copy of gfp-yhcS under the control of the pxyl promoter produced a largeramount of sortase than was produced from the wild-type sortasegene under the control of its own natural promoter in strainWB800 (Fig. 4A, lanes 6 and 7). Presumably, pxyl is a strongerpromoter than the endogenous sortase promoter. The productionlevel of GFP-YhcS (Fig. 4A, lanes 6 and 7) is much lower than theYhcS level from the strains carrying pE18-YhcS (Fig. 4A, lanes 3 to5). This is not unexpected, since the pE18 vector used in this studyhas a cop6 mutation that increases the copy number of this plas-mid in the host cell (61).

Quantification of the number of the wall-bound BLA-CWADYhcRMO2 in B. subtilis strains. To quantify the number ofBLA-CWADYhcRMO2 molecules that can be displayed on the cell




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FIG 4 Wall-bound BLA-CWADYhcRMO2 levels in relation to the cellular levels of YhcS and YhcS-GFP. (A) Comparison of sortase levels in different B. subtilisstrains by using Western blots probed with anti-YhcS antibodies. Cells of B. subtilis strains WB800, WB800Srt�, WB800(pE18-YhcS), WB800(pE18-YhcS,pWB980-BLA-CWADYhcRMO2), WB800Srt�(pE18-YhcS), WB800Srt� amyE::pxyl-gfp-yhcS, and WB800Srt� amyE:: pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) were treated with lysozyme and sonicated for immunoblotting. Samples from WB800Srt� (lane 1) and WB800 (lane 2) were concentrated sixtimes for this assay. As there were no discernible differences between 0.5 and 2.0% xylose induction, 1% xylose was used to induce GFP-YhcS throughout thisexperiment. The arrows indicate the GFP-YhcS produced by xylose induction. YhcS (21 kDa) is observed in lanes 3 to 5. The amounts of samples loaded werenormalized to cell density. Mouse anti-YhcS was used to probe for the reporter fusion with a 1:750 dilution. (B) Sortase overproduction increases the wall-bound

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surface, the amounts of BLA molecules anchored to the cellwall in B. subtilis WB800(pWB980-BLA-CWADYhcRMO2) andWB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2) were esti-mated by densitometric analysis of immunoblots (see Fig. S4Aand S4B in the supplemental material). The numbers of BLA-CWADYhcRMO2 molecules were determined to be 3,770 and47,300 molecules in strain WB800 and the sortase overproductionstrain, respectively (Fig. S4C). This observation indicates thathigher levels of BLA-CWADYhcRMO2 can be displayed by increas-ing the levels of sortase.

Functional activity of BLA-CWADYhcRMO2. To examinewhether the displayed BLA molecules are biologically active, theenzymatic activity of �-lactamase was measured by using wholecells from the following three strains: WB800(pWB980-BLA-CWADYhcRMO2), WB800(pWB980) (negative control), andWB800(pWB980-BLA), a strain that secretes BLA into the growthmedium. The background level in the absence of any �-lactamasewas also measured by measuring buffer without any cells added.Although extremely low activity (�5 units) was detected in eitherthe buffer or the washed cell fraction of the negative control(WB800 carrying the empty vector), enzymatic activity (�18units) from the washed cells of WB800(pWB980-BLA) was signif-icantly higher. This activity could likely be attributed to BLA mol-ecules trapped nonspecifically in the cell wall, as the �-lactamasesubstrate (PADAC) is small enough to diffuse into the peptidogly-can. In contrast, the enzymatic activity detected from strainWB800(pWB980-BLA-CWADYhcRMO2) was significantly higherwith 35 units. Both the wall-bound and membrane-bound formsof BLA-CWADYhcRMO2 can contribute to this observed activity.These data indicate that the �-lactamase reporter fused toCWADYhcRMO2 was biologically active.

DISCUSSION

Characterization of YhcR by Oussenko and coworkers (45) sug-gests that YhcR is a nuclease that may anchor to the cell wallcovalently. In this study, B. subtilis yhcS and the sequence encod-ing the cell wall anchoring domain of YhcR were characterized.This allows us to present conclusive evidence for the presence of abiologically functional sortase system in B. subtilis with YhcS asthe sortase and YhcR as the sortase substrate.

Spiral-like distribution of the sortase substrate YhcR. Immu-nofluorescence visualization of BLA-CWADYhcRMO2 on the sur-face of B. subtilis revealed that the sortase substrate was distributedin helices around the surface of the cell (Fig. 5). A similar obser-vation was also made by Nguyen and Schumann with their engi-neered B. subtilis surface display system (44). Why do BLA-CWADYhcRMO2 molecules distribute themselves in a helicalmanner? Previous studies of B. subtilis have suggested that nascentglycan strands are inserted helically (11, 28) into the lateral cellwall at several dispersed sites (1, 29, 54). During cell elongation,

these maturing helical glycan cables will develop a rotationaltorque, allowing it to sweep across the cell surface (6, 7). SinceBLA-CWADYhcRMO2 molecules are covalently anchored to thepeptidoglycan strands, it is not too unexpected to see the spiral-like distribution of BLA-CWADYhcRMO2.

Short helical arcs of YhcS and implications for cell wall bio-synthesis. As demonstrated in this study, the sorting of BLA-CWADYhcRMO2 to the cell wall is dependent on the catalytic activ-ity of YhcS. It would be of interest to examine the distribution ofYhcS for two reasons. First, sortases in cocci (Staphylococcus au-reus and Streptococcus pyogenes) are found to localize in specificmembrane foci (12, 47). However, the distribution of sortase(s) inrod-shaped bacteria has not been reported. Do sortases distributerandomly or in a highly organized manner? Second, are there anycorrelations between the distributions of the cell wall-anchoredsortase substrates and the sortase? Our results show that GFP-YhcS localizes in certain spatially restricted zones near the lateralcell wall (Fig. 6). Some formed short arcs, helices, or patches.Although the levels of GFP-YhcS detected were higher than thephysiological levels of YhcS, GFP-YhcS was not highly overpro-duced (Fig. 4A, lane 2 versus lanes 6 and 7). Therefore, the ob-served localization patterns were likely to represent the genuinephysiological distribution of sortase in B. subtilis. Some spots in ashort segment of the GFP-YhcS showed a certain degree of colo-calization with BLA-CWADYhcRMO2 (Fig. 7). These colocalizedsortases may be in the process of anchoring BLA-CWADYhcRMO2

to the peptidoglycan strand. We did not observe a long stretch ofoverlap between the BLA-CWADYhcRMO2 cables and the GFP-YhcS arcs. This may reflect the segregation of the BLA-CWADYhcRMO2 anchored peptidoglycan strands from the sorta-ses. Any initial colocalization would be lost after the transientinteractions between the maturing peptidoglycan strands and thesortase arcs during cell wall maturation and cell elongation pro-cesses.

The next notable question is how sortase, a membrane protein,would be localized to certain sites on the membrane without lat-eral diffusion. There are at least three possible models. For the firstmodel, the localization pattern of GFP-YhcS resembles the helicalpatterns previously observed for a number of proteins such as thecytoplasmic shape-determining cytoskeletal proteins MreB/Mbl/MreBH (13, 24, 27, 31), enzymes (e.g., penicillin binding proteins[PBPs] and teichoic acid biosynthetic enzymes) involved in cellwall synthesis (16, 24, 25, 49, 50) during cell elongation, and thescaffold membrane proteins, MreC and MreD (18, 21, 24, 34, 57),that act as a bridge between the cytoskeletal proteins and the cellwall synthesizing proteins. These proteins assemble to form mul-tienzyme complexes (known as holoenzymes) to mediate cell wallbiosynthesis. MreB, MreBH, and Mbl have been suggested to formcontinuous helical cytoskeletal cables located underneath the cellmembrane to guide the movement of the sidewall elongation ma-

BLA-CWADYhcRMO2 level. Strains WB800(pWB980-BLA-CWADYhcRMO2) (control) and WB800(pE18-YhcS, pWB980-BLA-CWADYhcRMO2) (sortase overpro-duction strain) were treated with lysozyme. The soluble supernatant fraction (S) of the lysozyme-treated samples and the protoplast fraction (P) are indicatedabove the gel. BLA-CWADYhcRMO2 levels in these fractions were analyzed by Western blotting. The membrane-bound form (M) (open circle) and the wall-boundform (W) (arrow) of BLA-CWADYhcRMO2 are indicated. The amounts of samples loaded were normalized to cell density. Rabbit anti-BLA was used to probe forthe reporter fusion with a 1:2,000 dilution. (C) Detection of BLA-CWADYhcRMO2 from the purified cell wall of B. subtilis WB800Srt� amyE::pxyl-gfp-yhcS. StrainWB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) was induced with 1% xylose. Purified cell wall samples from strains WB800(pWB980-BLA-CWADYhcRMO2) and WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) were used for Western blotting. The cell wall samples in lanes 1 and 2 weretreated with lysozyme to release BLA-CWADYhcRMO2, and the cell wall sample in lane 3 was not treated with lysozyme. The expected wall-bound form of BLA(filled arrowhead) and the wall-bound BLA anchored to the incompletely digested cell wall (open arrowheads) are indicated.




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FIG 5 Visualization of BLA-CWADYhcRMO2 on Bacillus subtilis WB800(pWB980-BLA-CWADYhcRMO2) cell surface by immunofluorescence microscopy. (A)Localization of BLA-CWADYhcRMO2 using an epifluorescence microscope. The cells were probed with rabbit anti-BLA antibodies (followed by Alexa Fluor488-conjugated anti-rabbit antibodies as the secondary antibody [green]), Alexa Fluor 594-conjugated wheat germ agglutinin (WGA) (red), and DAPI (blue).(a) A differential interference contrast (DIC) filter was applied to enhance the contrast of Bacillus subtilis cells for easier visualization. (b to d) Cells stained withDAPI, WGA, or anti-BLA antibodies, respectively. (e to h) Composite images generated by merging images obtained from panels a to d. (B) Application of thedeconvolution software to reduce the optical distortion and noise obtained in the epifluorescence microscope image. The image used for this application was thesame cell as shown in Fig. 4A. (a to d) Composite images showing the localization of BLA-CWADYhcRMO2 with respect to DNA and cell wall (WGA). (C) Z-stackimages of BLA-CWADYhcRMO2 through the same cell in panel A. Eight planes of focus are shown. The red arrows indicate helical filaments observable throughslices 3 to 8 (from gradually visible in slices 1 to 3 to optimally visible in slices 4 to 6 to gradually invisible in slices 7 and 8). The blue arrows indicate helicalfilaments observable through slices 1 to 6. Illustrations showing the current optical focus of the microscope used to observe the different planes of the cell is shown

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chineries (including MreC, MreD, RodA, RodZ, and cell wall bio-synthetic enzymes such as PBPs) along the sidewall. As sortase isresponsible for covalently anchoring cell wall proteins, it is tempt-ing to speculate that YhcS is associated with the cell wall biosyn-thetic machinery. This can account for the observed distributionof YhcS. Interestingly, new techniques, including the total internalreflection fluorescence microscopy and cryoelectron tomography

(19, 26, 56) have recently been applied to study the dynamic dis-tribution of B. subtilis MreB and other components in the sidewallelongation machineries during cell elongation. These findingssuggest that MreB, MreBH, and Mbl form short segments orpatches which exert a circumferential motion along the cell pe-riphery. The sidewall biosynthetic enzyme complexes also have asimilar dynamic motion. Because of the progressive motion of

on the upper right corner. (D) WB800(pWB980) cells stained with DAPI for DNA (blue in panel b), Alexa Fluor 594-conjugated wheat germ agglutinin (WGA)(red in panel c), and rabbit anti-BLA antibodies (followed by Alexa Fluor 488-conjugated goat anti-rabbit antibodies, green in panel d). (e to g) Composite imagesgenerated by merging images obtained from panels a to d. (E) WB800(pWB980-BLA-CWADYhcRMO2) cells probed with Alexa Fluor 488-conjugated antibodiesagainst rabbit anti-BLA antibodies (green) only without preprobing with the anti-BLA antibodies. (a) DIC image; (b) cells observed with blue, green, and redfilters; (c) composite image generated by merging images obtained by using DAPI, WGA, and Alexa Fluor 488.

FIG 6 GFP-YhcS distribution as observed by fluorescence microscopy. (A) Localization of GFP-YhcS in B. subtilis WB800Srt� amyE::pxyl-gfp-yhcS cultivated inSRM supplemented with 1% xylose. (a) Differential interference contrast (DIC) filter to enhance the contrast in Bacillus subtilis cells for easier visualization. (b)Observation of the same cells viewed with a GFP filter with deconvolution. As z-stacks were obtained for deconvolution, only one optical plane of focus ispresented here in panel b. The cell highlighted in the white box was used to show the intertwining ribbons observed under different optical sections (in panel B).(B) Intertwining ribbons of GFP-YhcS discernible by assembling a series of z-stack images (a to f) taken in successive planes following by sharpening usingdeconvolution. Optical sections of the cell from panel A (white box) was used for deconvolution. Six xz planes were used to show the development of theintertwining ribbons as the image is moved downward in the z plane. An illustration showing the current optical plane of focus used to observe GFP-YhcS in thecell is shown in the top right corner. (Inset in panel c) Illustration showing the direction of ribbons observed in panel c. (C) Helices observed in strain WB800Srt�

amyE::pxyl-gfp-yhcS through z-stack images. The cell observed here is a different cell from the one shown in panels A and B. The cells were grown overnight in SRMsupplemented with 1% xylose. An illustration showing the current optical plane of focus used to observe GFP-YhcS in the cell is shown in the top right corner.(a) Illustration showing the half-helix observed in the images in panels b to f. (b to f) Z-stack images demonstrating the development of a half-helix as the opticalsection is moved downwards in the xz plane. The white arrows indicate the development of half-helix through slices 1 to 5. (D) Distribution of GFP in strainWB800Srt� amyE::pxyl-gfp. WB800Srt� amyE::pxyl-gfp cells were grown overnight in SRM supplemented with 1% xylose. The cells were washed twice with PBSbefore they were spotted onto a microscope slide and observed using the DIC filter and the GFP filter.




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FIG 7 Colocalization of BLA-CWADYhcRMO2 with GFP-YhcS observed by fluorescence microscopy. (A) Localization of DNA, BLA-CWADYhcRMO2, andGFP-YhcS using an epifluorescence microscope. WB800Srt� amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) cells were probed with rabbit anti-BLA anti-bodies (followed by Alexa Fluor 568-conjugated goat anti-rabbit antibodies as the secondary antibody) and DAPI. (a to c) Observation of strain WB800Srt�

amyE::pxyl-gfp-yhcS(pWB980-BLA-CWADYhcRMO2) with different filters. DNA was observed under the DAPI (blue) filter, BLA-CWADYhcRMO2 was observedunder the Alexa Fluor 568 (red) filter, and GFP-YhcS was observed with the GFP filter (green). (B) Z-stacks of a composite image generated by merging individualimages obtained from panel A under DAPI (blue), GFP (green), and Alexa Fluor 568 (red) filters. Each composite image represents one optical section of the xzplane as the plane is shifted downwards (panels a to f). Red arrows indicate spots of colocalization (yellow) of BLA-CWADYhcRMO2 and GFP-YhcS seen byoverlapping of red- and green-colored regions. White arrowheads indicate the regions where colocalization will develop when the z plane of view is moveddownwards through the cell. (Inset panel a) Cartoon illustrating the observed colocalization spots (yellow) which appear progressively in z planes a to f. Yellowspots 1 to 4 in the cartoon correspond to arrows 1 to 4 in the panel.

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these MreB patches along the cell periphery and the increaseddepth of field in epifluorescence microscopy, MreB and compo-nents in the sidewall biosynthetic machinery could easily be con-sidered to form continuous helical cables in previous studies (6,24, 27, 31). In our current study, GFP-sortases were found to formshort helical arcs in most cases. If YhcS is part of the sidewallbiosynthetic enzyme complex, this observation can be explainedwell by the dynamic circumferential motion model.

For the second model, structures other than the cell wall bio-synthetic complexes could also influence the helical distributionof YhcS. Recently, Barák and colleagues (3) have demonstratedthat lipid spirals extend along the axis of B. subtilis and that the celldivision protein MinD, which is attached to the lipid spirals, alsoforms spiral structures. Similarly, if YhcS attaches to these spirallydistributed lipids, YhcS may distribute helically. Last, the Sec-dependent secretion apparatus in both B. subtilis and E. coli wasshown to be distributed helically (5, 52). Evidence for the colocal-ization of sortase and the Sec apparatus in Streptococcus mutanshas also been presented (30). It is possible that YhcS moleculesreleased from the helically distributed Sec apparatus in B. subtilisdistribute themselves in a helical manner. In this case, it is unclearhow these short helical arcs of YhcS can maintain their distribu-tion in the membrane without lateral diffusion. A combination ofthese models may also be possible.

Quantification of the wall-bound BLA-CWADYhcR. Due tothe small molecular mass difference, a clean separation of the wall-bound form of BLA-CWADYhcR from the membrane-bound/pre-cursor forms of the reporter fusion by SDS-PAGE would be diffi-cult. As a consequence, it was impossible to conclude that a yhcSknockout strain has been successfully constructed (compare Fig.1D, lanes 1 and 2, to Fig. 3, lanes 1 and 3) and that YhcS is respon-sible for anchoring YhcR to the cell wall. Furthermore, the amountof BLA-CWADYhcR covalently linked to the cell wall would beoverestimated by at least four- to fivefold (Fig. 1D, lanes 1 and 3).Our approach to overcome these problems by fusing a mono-meric protein to the C-terminal tail of BLA-CWADYhcR can beuniversally applied to other sortase systems if serious quantifica-tion of the amounts of covalently wall-bound proteins is needed.The number of wall-bound BLA-CWADYhcRMO2 in this system isapproximately 5 times lower than that of the display system re-ported by Nguyen and Schumann (44). This difference can beattributed to different factors. The system developed by Nguyenand Schumann is based on the sortase from Listeria monocyto-genes, the cell wall anchoring domain from the S. aureus fibronec-tin binding protein B, and the �-amylase reporter from Bacillusamyloliquefaciens. The ability of the wall-bound fusion protein tobe resistant to the protease activity from B. subtilis is an importantfactor in determining the number of molecules that can be suc-cessfully displayed. Protease resistance depends on the nature ofthe domains used in the fusion construction and the length of thelinker. Linkers that are too short (94 amino acids) or too long (162or more amino acids) can reduce the number of molecules that aredisplayed on the cell surface (44). It is possible that differentsortase-based display systems can have a difference in the numberof molecules that can be displayed on the surface. The other pos-sible reason may be due to the use of the MO2 fusion approach inthis study.

Development of a robust cell surface display system. Weshowed that our reporter fusions are functional and surface acces-sible. The effective pore radius of the B. subtilis peptidoglycan is

estimated to be �2.1 nm (15). Under these conditions, only glob-ular proteins with molecular masses less than 50 kDa are predictedto be able to penetrate through the peptidoglycan layers. The im-munoglobulin G molecules used in this study for the probing ofthe surface-displayed �-lactamase have molecular masses in therange of 150 kDa. Consequently, they should not be able to pen-etrate through the peptidoglycan layers. This assumption, in fact,has been demonstrated to be the case in Streptococcus pyogenes(47). Therefore, successful probing of �-lactamase by IgG reflectsthe surface accessibility of the wall-bound proteins. Successful dis-play of functional enzymes to the microbial cell surface showsincreasing promise in developing whole-cell biocatalysts, as oursociety progresses toward an industrialized and technologicallydependent future (60). However, the major challenge of a micro-bial surface display at the moment is the development of a systemthat is sufficiently robust for industrial applications (4, 32, 37, 48).Fragility of the outer membrane of Gram-negative bacteria (e.g.,E. coli) can be a major concern for surface display. This problemcan potentially be circumvented in Gram-positive bacteria withthe use of the cell wall as the display platform. There are at leasttwo advantages of the YhcS-YhcR-based surface display system.First, in contrast to the noncovalent surface display systems usingthe autolysin-based cell wall binding domains for surface display,the CWADYhcR fusions are covalently anchored to the cell surfaceand will not be easily detached from the cell surface even in anenvironment with a high external shearing force. Second, the dis-played molecules are distributed along the lateral cell wall and notconcentrated at the septa. This is a distinct advantage compared toother display systems that are localized at the septa (43). Display ofproteins along the lateral cell wall should allow optimal exposureof the displayed enzymes toward the substrate. Furthermore, thissystem which displays moderate levels of molecules (104 mole-cules per cell) on the cell surface can potentially be more appro-priate for displaying giant enzyme complexes such as cellulo-somes. The molecular masses of cellulosomes can be �2 � 106 Daor greater (53). Each cellulosome is composed of tens of subunits.Relative to other high-density surface display systems that display106 to 107 molecules per cell (9, 33), this system may have a betterchance to allow the displayed complexes to have sufficient room tointeract with their binding targets without imposing steric hin-drance between complexes.

Other putative B. subtilis sortase substrates and sortases.YfkN, a 2=,3= cyclic nucleotide phosphodiesterase (8), is the otherputative sortase substrate with an LPDTA motif, a transmem-brane segment, and a positively charged C-terminal tail (8). Sev-eral BLA-CWADYfkN fusions were constructed. However, theCWADYfkN domain seemed to be very susceptible to the residualproteases in B. subtilis WB800. Convincing evidence for theseBLA-CWADYfkN fusions as the covalently linked wall-bound pro-teins could not be obtained. Recently, an enzyme known asLPXTGase which competes with sortase to cleave sortase sub-strates in S. pyogenes and S. aureus has been identified (35, 36).Since the sequence of this enzyme is unknown, it is not clearwhether there is a B. subtilis LPXTGase-like enzyme that has apreference to cleave YfkN.

YwpE is the second putative sortase in B. subtilis. The openreading frame for ywpE, in fact, encodes a truncated sortase-likeprotein. The AUG codon annotated in the SubtiList web server asthe initiation codon for this gene is incorrect for two reasons. First,there is no ribosome binding site located upstream of this codon.




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Even though the mRNA for this gene can be translated, the trans-lated product will not have an N-terminal transmembrane seg-ment to anchor this protein to the membrane as a functional sor-tase. Second, the suggested N-terminal methionine residue forYwpE is, in fact, a conserved internal methionine in the sortasefamily. If the coding sequence is extended upstream of this methi-onine codon, the 28 amino acid residues located immediately up-stream of this so-called N-terminal methionine residue can alignvery well with the conserved sortase sequences. These findingssuggest that ywpE is an incomplete gene. B. subtilis WB800 is aderivative of B. subtilis 168. Alignment of B. subtilis 168 YwpE withthe translated gene products from the genomes of B. subtilis subsp.spizizenii strain W23, B. subtilis ATCC 6633, Bacillus licheniformis,and B. amyloliquefaciens indicates that there is a YwpE homolog ineach of these strains and species. In reference to these YwpE ho-mologs, the B. subtilis 168 version of YwpE misses the first 81- or82-amino-acid sequence which includes the N-terminal trans-membrane segment. Interestingly, the gene next to ywpE in B.subtilis subsp. spizizenii strain W23, B. subtilis ATCC 6633, B. li-cheniformis, and B. amyloliquefaciens encodes a protein with sim-ilarity to collagen binding proteins. This protein has a putativesortase-mediated cell wall anchoring motif. YwpE may be respon-sible for anchoring this putative collagen binding protein to thecell surface. In B. subtilis 168, this collagen binding protein geneand the coding sequence for the beginning of YwpE are absent. Itis possible that this gene segment was deleted during evolution,and the ywpE gene in B. subtilis 168 is likely to be defective.

What is the rationale of immobilizing YhcR and YfkN to thecell surface if these proteins are released into the culture super-natant by proteases from B. subtilis? Although YfkN cannot beshown to be a covalently linked wall-bound protein in this study,a recent paper (22) provides evidence that YfkN is a covalentlylinked wall-bound protein. In the aforementioned study, theamount of YfkN in the culture supernatant produced from a sor-tase mutant cultivated under phosphate starvation condition istwo times higher than that produced from the wild-type strain. Itis hypothesized that anchoring YfkN to the cell wall minimizes theproteolytic cleavage of the membrane-bound form of YfkN by B.subtilis proteases. Since both YhcR and YfkN can be detected in theculture medium (8, 22, 45), this observation poses an interestingquestion concerning the purpose of immobilizing these enzymeson the cell surface. Although there is no definite answer to thisquestion, the presence of YhcR and YfkN in both the wall-boundforms and the proteolytic cleaved forms in the culture supernatantcan possibly be considered a very elegant strategy employed by B.subtilis. Under phosphate starvation conditions, cells would like tooptimize the local phosphate uptake from the immediate sur-rounding environment as much as possible. B. subtilis might beable to use its cell wall as a scaffold or platform to concentrate andlocalize YhcR and YfkN. Wall-bound YhcR initiates the cleavageof any available RNA from the local environment into nucleotides.YfkN nucleotidases which are in close proximity can subsequentlycapture these locally released 2=, 3=, or 5= nucleotides and hydro-lyze them to releases phosphates which can now be easily im-ported into the cells. Since colocalization of these enzymes on thecell surface can potentially allow RNA to be hydrolyzed to phos-phates efficiently for subsequent uptake, it would be important tohave these newly synthesized nucleases and nucleotidases immo-bilized to the cell surface at least transiently. In addition, the com-bined actions of both YhcR and YfkN in the culture medium allow

the release of phosphates from RNA present in the environment.B. subtilis would be able to salvage phosphates from RNA whetherthese RNA molecules are far away from the cells or close to the cellsurface. With the physiological functions of YhcR and YfkN inmind, the low YhcS activity and the inability to detect YhcS byWestern blotting when cells were cultivated in a phosphate-richsuperrich medium can potentially be explained. The YhcS levelmay be higher when cells are phosphate starved (22).

ACKNOWLEDGMENTS

We thank Lorie Kwang for the construction of pWB980-BLA-CWADYhcR, Chyi-Liang Chen for the purified �-lactamase, Dave Hansenfor the use of the Carl Zeiss Axioimager Z1 fluorescence compound mi-croscope, Xiao-Zhou Zhang from Virginia Polytechnic Institute and StateUniversity for the recommendation of the marker-free gene knockoutsystem used in this study, Peter J. Lewis from the University of Newcastlefor the helpful discussions of some technical aspects of using pSG1729,and the Bacillus Genetic Stock Center for pSG1729, p7S6, and pTSC.

The research work is supported by a discovery grant from the NaturalSciences and Engineering Research Council of Canada (NSERC).

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